It is now well known that cells can be infected by HIV through a process by which fusion occurs between the cellular membrane and the viral membrane. The generally accepted model of this process is that the viral envelope glycoprotein complex (gp120/gp41) interacts with cell surface receptors on the membranes of the target cells. Following binding of gp120 to cellular receptors (e.g., CD4 in combination with a chemokine co-receptor such as CCR-5 or CXCR-4), a conformational change is induced in the gp120/gp41 complex that allows gp41 to insert into the membrane of the target cell and mediate membrane fusion.
The amino acid sequence of gp41, and its variation among different strains of HIV, is well known. FIG. 1 is a schematic representation of the generally accepted functional domains of gp41 (note the amino acid sequence numbers may vary slightly depending on the HIV strain). The fusogenic domain is believed to be involved in insertion into and disruption of the target cell membrane. The transmembrane domain, containing the transmembrane anchor sequence, is located at the C-terminal end of the protein. Between the fusogenic domain and transmembrane anchor are two distinct regions, known as heptad repeat (HR) regions, each region having a plurality of heptads. The amino acid sequence comprising the HR1 region and the amino acid sequence comprising the HR2 region are each highly conserved regions in the HIV-1 envelope protein. The HR2 region has been generally described as comprising amino acid residues of SEQ ID NO:1, or polymorphisms thereof (see, e.g., FIG. 2). As further shown in FIG. 1, the HR regions have a plurality of 7 amino acid residue stretches or “heptads” (the 7 amino acids in each heptad designated “a” through “g”), with a predominance of hydrophobic residues at the first (“a”) and fourth (“d”) positions, and charged residues frequently at the fifth (“e”) and seventh (“g”) positions. Also present in the amino acid sequence of HIV gp41 are leucine zipper-like motifs comprising an 8 amino acid sequence initiating with, and ending with, either an isoleucine or leucine. As illustrated in FIG. 1, the HR2 region has just one leucine zipper like-motif, whereas the HR1 region has five leucine zipper-like motifs. The heptads and a leucine zipper-like motif are amino acid sequence features thought to contribute to formation of the coiled coil structure found for gp41.
It was discovered that peptides derived from the natural sequence of either the HR1 region (“HR1 peptides”) or HR2 region (“HR2 peptides”) of HIV gp41 inhibit transmission of HIV to host cells both in in vitro assays and in in vivo clinical studies. For example, HR2 peptides, as exemplified by DP178 (also known as T20, enfuvirtide, and Fuzeon®; SEQ ID NO:2), T651 (SEQ ID NO:3), T649 (SEQ ID NO:4), blocked infection of target cells with potencies of 0.5 ng/ml (EC50 against HIV-1LA1), 5 ng/ml (IC50; HIV-1 IIIB), and 2 ng/ml (IC50; HIV-1 IIIB), respectively. Efforts have been made to improve the biological activity of HIV gp41-derived peptides, such as by trying to stabilize the helical structure of the peptide. For example, synthetic peptides having helix stabilization are disclosed by the present inventors in PCT publication WO 2005/067960, and are exemplified as SEQ ID NOs:5-7 herein. Synthetic peptides which can inhibit HIV fusion (a process by which HIV gp41 mediates fusion between the viral membrane and the membrane of the cell during the infection process by HIV of a target cell) are a class of peptides often referred to as HIV fusion inhibitor peptides.
Another drawback associated with synthetic peptides relates to the solubility and stability in aqueous-based pharmaceutically acceptable carriers, such as relating to the process of making an injectable solution formulation of an HIV fusion inhibitor peptide. For example, it is difficult to achieve an injectable aqueous solution containing a synthetic peptide having an amino acid sequence of SEQ ID NO:2 in a concentration of more than 100 mg/ml without encountering problems of solubility (wherein the formulation resembles a gel, rather than a solution, or peptide precipitates out of solution over a predetermined time period) and stability (peptide being degraded over a predetermined period of time), and without adding additional components to the formulation to promote stability and/or solubility. Also it would be desirable to develop an HIV fusion inhibitor peptide having improved solubility and stability, while also having improved pharmacological properties.
Thus, there is a need for an HIV fusion inhibitor peptide which: (a) when added in an effective amount, can interfere with the viral fusion process mediated by HIV gp41, and more preferably, interfere with the conformational changes of gp41 necessary to effect fusion, thereby inhibiting the fusion of HIV gp41 to a target cell membrane; (b) demonstrates improved solubility and stability in an aqueous solution; and (c) demonstrates improved pharmacological properties. The present invention addresses these needs.